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September 22, 2019

Nuclear and mitochondrial genomes of the hybrid fungal plant pathogen Verticillium longisporum display a mosaic structure

Allopolyploidization, genome duplication through interspecific hybridization, is an important evolutionary mechanism that can enable organisms to adapt to environmental changes or stresses. This increased adaptive potential of allopolyploids can be particularly relevant for plant pathogens in their quest for host immune response evasion. Allodiploidization likely caused the shift in host range of the fungal pathogen plant Verticillium longisporum, as V. longisporum mainly infects Brassicaceae plants in contrast to haploid Verticillium spp. In this study, we investigated the allodiploid genome structure of V. longisporum and its evolution in the hybridization aftermath. The nuclear genome of V. longisporum displays a mosaic structure, as numerous contigs consists of sections of both parental origins. V. longisporum encountered extensive genome rearrangements, whereas the contribution of gene conversion is negligible. Thus, the mosaic genome structure mainly resulted from genomic rearrangements between parental chromosome sets. Furthermore, a mosaic structure was also found in the mitochondrial genome, demonstrating its bi-parental inheritance. In conclusion, the nuclear and mitochondrial genomes of V. longisporum parents interacted dynamically in the hybridization aftermath. Conceivably, novel combinations of DNA sequence of different parental origin facilitated genome stability after hybridization and consecutive niche adaptation of V. longisporum.

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September 22, 2019

Complete genome sequence of Pseudomonas Parafulva PRS09-11288, a biocontrol strain produces the antibiotic phenazine-1-carboxylic acid.

Rhizoctonia solani is a plant pathogenic fungus, which can infect a wide range of economic crops including rice. In this case, biological control of this pathogen is one of the fundmental way to effectively control this pathogen. The Pseudomonas parafulva strain PRS09-11288 was isolated from rice rhizosphere and shows biocontrol ability against R. solani. Here, we analyzed the P. parafulva genome, which is ~?4.7 Mb, with 4310 coding sequences, 76 tRNAs, and 7 rRNAs. Genome analysis identified a phenazine biosynthetic pathway, which can produce antibiotic phenazine-1-carboxylic acid (PCA). This compound is responsible for biocontrol ability against R. solani Kühn, which is one of the most serious fungus disease on rice. Analysis of the phenazine biosynthesis gene mutant, ?phzF, which is very important in this pathway, confirmed the relationship between the pathway and PCA production using LC-MS profiles. The annotated full genome sequence of this strain sheds light on the role of P. parafulva PRS09-11288 as a biocontrol bacterium.

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September 22, 2019

Microbiome and infectivity studies reveal complex polyspecies tree disease in Acute Oak Decline.

Decline-diseases are complex and becoming increasingly problematic to tree health globally. Acute Oak Decline (AOD) is characterized by necrotic stem lesions and galleries of the bark-boring beetle, Agrilus biguttatus, and represents a serious threat to oak. Although multiple novel bacterial species and Agrilus galleries are associated with AOD lesions, the causative agent(s) are unknown. The AOD pathosystem therefore provides an ideal model for a systems-based research approach to address our hypothesis that AOD lesions are caused by a polymicrobial complex. Here we show that three bacterial species, Brenneria goodwinii, Gibbsiella quercinecans and Rahnella victoriana, are consistently abundant in the lesion microbiome and possess virulence genes used by canonical phytopathogens that are expressed in AOD lesions. Individual and polyspecies inoculations on oak logs and trees demonstrated that B. goodwinii and G. quercinecans cause tissue necrosis and, in combination with A. biguttatus, produce the diagnostic symptoms of AOD. We have proved a polybacterial cause of AOD lesions, providing new insights into polymicrobial interactions and tree disease. This work presents a novel conceptual and methodological template for adapting Koch’s postulates to address the role of microbial communities in disease.

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September 22, 2019

Characterization of ß-glucan formation by Lactobacillus brevis TMW 1.2112 isolated from slimy spoiled beer.

Despite several hurdles, which hinder bacterial growth in beer, certain bacteria are still able to spoil beer. One type of spoilage is characterized by an increased viscosity and slimy texture caused by exopolysaccharide (EPS) formation of lactic acid bacteria (LAB). In this study, we characterize for the first time EPS production in a beer-spoiling strain (TMW 1.2112) of Lactobacillus brevis, a species commonly involved in beer spoilage. The strain’s growth dynamics were assessed and we found an increased viscosity or ropiness in liquid or on solid media, respectively. Capsular polysaccharides (CPS) and released EPS from the cells or supernatant, respectively, were analyzed via NMR spectroscopy and methylation analysis. Both are identical ß-(1?3)-glucans, which are ramified with ß-glucose residues at position O2. Therefore, we assume that this EPS is mainly produced as CPS and partially released into the surrounding medium, causing viscosity of e.g. beer. CPS formation was confirmed via an agglutination test. A plasmid-located glycosyltransferase-2 was found as responsible for excess ß-glucan formation, chromosomal glucanases were proposed for its degradation. The glycosyltransferase-2 gene could also be specifically identified in beer-spoiling, slime-producing Lactobacillus rossiae and Lactobacillus parabuchneri strains, suggesting it as promising marker gene for the early detection of ß-glucan-producing Lactobacilli in breweries. Copyright © 2017 Elsevier B.V. All rights reserved.

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September 22, 2019

The repeat structure of two paralogous genes, Yersinia ruckeri invasin (yrInv) and a “Y. ruckeri invasin-like molecule”, (yrIlm) sheds light on the evolution of adhesive capacities of a fish pathogen.

Inverse autotransporters comprise the recently identified type Ve secretion system and are exemplified by intimin from enterohaemorrhagic Escherichia coli and invasin from enteropathogenic Yersiniae. These proteins share a common domain architecture and promote bacterial adhesion to host cells. Here, we identified and characterized two putative inverse autotransporter genes in the fish pathogen Yersinia ruckeri NVH_3758, namely yrInv (for Y. ruckeri invasin) and yrIlm (for Y. ruckeri invasin-like molecule). When trying to clone the highly repetitive genes for structural and functional studies, we experienced problems in obtaining PCR products. PCR failures and the highly repetitive nature of inverse autotransporters prompted us to sequence the genome of Y. ruckeri NVH_3758 using PacBio sequencing, which produces some of the longest average read lengths available in the industry at this moment. According to our sequencing data, YrIlm is composed of 2603 amino acids (7812bp) and has a molecular mass of 256.4kDa. Based on the new genome information, we performed PCR analysis on four non-sequenced Y. ruckeri strains as well as the sequenced. Y. ruckeri type strain. We found that the genes are variably present in the strains, and that the length of yrIlm, when present, also varies. In addition, the length of the gene product for all strains, including the type strain, was much longer than expected based on deposited sequences. The internal repeats of the yrInv gene product are highly diverged, but represent the same bacterial immunoglobulin-like domains as in yrIlm. Using qRT-PCR, we found that yrIlm and yrInv are differentially expressed under conditions relevant for pathogenesis. In addition, we compared the genomic context of both genes in the newly sequenced Y. ruckeri strain to all available PacBio-sequenced Y. ruckeri genomes, and found indications of recent events of horizontal gene transfer. Taken together, this study demonstrates and highlights the power of Single Molecule Real-Time technology for sequencing highly repetitive proteins, and sheds light on the genetic events that gave rise to these highly repetitive genes in a commercially important fish pathogen. Copyright © 2017 Elsevier Inc. All rights reserved.

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September 22, 2019

Comparative genomics and transcriptomics analysis-guided metabolic engineering of Propionibacterium acidipropionici for improved propionic acid production.

Acid stress induced by the accumulation of organic acids during the fermentation of propionibacteria is a severe limitation in the microbial production of propionic acid (PA). To enhance the acid resistance of strains, the tolerance mechanisms of cells must first be understood. In this study, comparative genomic and transcriptomic analyses were conducted on wild-type and acid-tolerant Propionibacterium acidipropionici to reveal the microbial response of cells to acid stress during fermentation. Combined with the results of previous proteomic and metabolomic studies, several potential acid-resistance mechanisms of P. acidipropionici were analyzed. Energy metabolism and transporter activity of cells were regulated to maintain pH homeostasis by balancing transmembrane transport of protons and ions; redundant protons were eliminated by enhancing the metabolism of certain amino acids for a relatively stable intracellular microenvironment; and protective mechanism of macromolecules were also induced to repair damage to proteins and DNA by acids. Transcriptomic data indicated that the synthesis of acetate and lactate were undesirable in the acid-resistant mutant, the expression of which was 2.21-fold downregulated. In addition, metabolomic data suggested that the accumulation of lactic acid and acetic acid reduced the carbon flow to PA and led to a decrease in pH. On this basis, we propose a metabolic engineering strategy to regulate the synthesis of lactic acid and acetic acid that will reduce by-products significantly and increase the PA yield by 12.2% to 10.31?±?0.84?g/g DCW. Results of this study provide valuable guidance to understand the response of bacteria to acid stress and to construct microbial cell factories to produce organic acids by combining systems biology technologies with synthetic biology tools.© 2017 Wiley Periodicals, Inc.

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September 22, 2019

The putative functions of lysogeny in mediating the survivorship of Escherichia coli in seawater and marine sediment.

Escherichia coli colonizes various body parts of animal hosts as a commensal and a pathogen. It can also persist in the external environment in the absence of fecal pollution. It remains unclear how this species has evolved to adapt to such contrasting habitats. Lysogeny plays pivotal roles in the diversification of the phenotypic and ecologic characters of E. coli as a symbiont. We hypothesized that lysogeny could also confer fitness to survival in the external environment. To test this hypothesis, we used the induced phages of an E. coli strain originating from marine sediment to infect a fecal E. coli strain to obtain an isogenic lysogen of the latter. The three strains were tested for survivorship in microcosms of seawater, marine sediment and sediment interstitial water as well as swimming motility, glycogen accumulation, biofilm formation, substrate utilization and stress resistance. The results indicate that lysogenic infection led to tractable changes in many of the ecophysiological attributes tested. Particularly, the lysogen had better survivorship in the microcosms and had a substrate utilization profile resembling the sediment strain more than the wild type fecal strain. Our findings provide new insights into the understanding of how E. coli survives in the natural environment.© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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September 22, 2019

Complete genome sequence of Bacillus velezensis 157 isolated from Eucommia ulmoides with pathogenic bacteria inhibiting and lignocellulolytic enzymes production by SSF.

Bacillus velezensis 157 was isolated from the bark of Eucommia ulmoides, and exhibited antagonistic activity against a broad spectrum of pathogenic bacteria and fungi. Moreover, B. velezensis 157 also showed various lignocellulolytic activities including cellulase, xylanase, a-amylase, and pectinase, which had the ability of using the agro-industrial waste (soybean meal, wheat bran, sugarcane bagasse, wheat straw, rice husk, maize flour and maize straw) under solid-state fermentation and obtained several industrially valuable enzymes. Soybean meal appeared to be the most efficient substrate for the single fermentation of B. velezensis 157. Highest yield of pectinase (19.15 ± 2.66 U g-1), cellulase (46.69 ± 1.19 U g-1) and amylase (2097.18 ± 15.28 U g-1) was achieved on untreated soybean meal. Highest yield of xylanase (22.35 ± 2.24 U g-1) was obtained on untreated wheat bran. Here, we report the complete genome sequence of the B. velezensis 157, composed of a circular 4,013,317 bp chromosome with 3789 coding genes and a G + C content of 46.41%, one circular 8439 bp plasmid and a G + C content of 40.32%. The genome contained a total of 8 candidate gene clusters (bacillaene, difficidin, macrolactin, butirosin, bacillibactin, bacilysin, fengycin and surfactin), and dedicates over 15.8% of the whole genome to synthesize secondary metabolite biosynthesis. In addition, the genes encoding enzymes involved in degradation of cellulose, xylan, lignin, starch, mannan, galactoside and arabinan were found in the B. velezensis 157 genome. Thus, the study of B. velezensis 157 broadened that B. velezensis can not only be used as biocontrol agents, but also has potentially a wide range of applications in lignocellulosic biomass conversion.

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September 22, 2019

Insights on a founder effect: the case of Xylella fastidiosa in the Salento area of Apulia, Italy

Xylella fastidiosa causing disease on different plant species has been reported in several European countries, since 2013. Based on multilocus sequence typing (MLST) results, there is evidence of repeated introductions of the pathogen in Spain and France. In contrast, in the Salento area of Apulia (Puglia) in Southern Italy, the existence of a unique Apulian MLST genotype of X. fastidiosa, causing the olive quick decline syndrome (OQDS; also referred to as “CoDiRO” or “ST53”) was proven, and this was tentatively ascribed to X. fastidiosa subsp. pauca. In order to acquire information on intra population diversity European Food Safety Authority (EFSA) has strongly called for the characterization of X. fastidiosa isolates from Apulia to produce the necessary data to better understand strain diversity and evolution. In this work, for the first time the existence of sub-variants within a set of 14 “ST53” isolates of X. fastidiosa collected from different locations was searched using DNA typing methods targeting the whole pathogen genome. Invariably, VNTR, RAPD and rep-PCR (ERIC and BOX motifs) analyses indicated that all tested isolates possessed the same genomic fingerprint, supporting the existence of predominant epidemiological strain in Apulia. To further explore the degree of clonality within this population, two isolates from two different Salento areas (Taviano and Ugento) were completely sequenced using PacBio SMRT technology. The whole genome map and sequence comparisons revealed that both isolates are nearly identical, showing less than 0.001% nucleotide diversity. However, the complete and circularized Salento-1 and Salento-2 genome sequences were different, in genome and plasmid size, from the reference strain 9a5c of X. fastidiosa subsp. pauca (from citrus), and showed a PCR-proved large genome inversion of about 1.7 Mb. Genome-wide indices ANIm and dDDH indicated that the three isolates of X. fastidiosa from Salento (Apulia, Italy), namely Salento-1, Salento-2, and De Donno, whose complete genome sequence has been recently released, share a very recent common ancestor. This highlights the importance of continuous and extensive monitoring of molecular variation of this invasive pathogen to understand evolution of adaptive traits, and the necessity for adoption of all possible measures to reduce the risk of new introductions that may augment pathogen diversity.

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September 22, 2019

Cultivation-independent and cultivation-dependent analysis of microbes in the shallow-sea hydrothermal system off Kueishantao island, Taiwan: Unmasking heterotrophic bacterial diversity and functional capacity.

Shallow-sea hydrothermal systems experience continuous fluctuations of physicochemical conditions due to seawater influx which generates variable habitats, affecting the phylogenetic composition and metabolic potential of microbial communities. Until recently, studies of submarine hydrothermal communities have focused primarily on chemolithoautotrophic organisms, however, there have been limited studies on heterotrophic bacteria. Here, fluorescence in situ hybridization, high throughput 16S rRNA gene amplicon sequencing, and functional metagenomes were used to assess microbial communities from the shallow-sea hydrothermal system off Kueishantao Island, Taiwan. The results showed that the shallow-sea hydrothermal system harbored not only autotrophic bacteria but abundant heterotrophic bacteria. The potential for marker genes sulfur oxidation and carbon fixation were detected in the metagenome datasets, suggesting a role for sulfur and carbon cycling in the shallow-sea hydrothermal system. Furthermore, the presence of diverse genes that encode transporters, glycoside hydrolases, and peptidase indicates the genetic potential for heterotrophic utilization of organic substrates. A total of 408 cultivable heterotrophic bacteria were isolated, in which the taxonomic families typically associated with oligotrophy, copiotrophy, and phototrophy were frequently found. The cultivation-independent and -dependent analyses performed herein show that Alphaproteobacteria and Gammaproteobacteria represent the dominant heterotrophs in the investigated shallow-sea hydrothermal system. Genomic and physiological characterization of a novel strain P5 obtained in this study, belonging to the genus Rhodovulum within Alphaproteobacteria, provides an example of heterotrophic bacteria with major functional capacity presented in the metagenome datasets. Collectively, in addition to autotrophic bacteria, the shallow-sea hydrothermal system also harbors many heterotrophic bacteria with versatile genetic potential to adapt to the unique environmental conditions.

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September 22, 2019

Complete genome sequence and analysis of the industrial Saccharomyces cerevisiae strain N85 used in Chinese rice wine production.

Chinese rice wine is a popular traditional alcoholic beverage in China, while its brewing processes have rarely been explored. We herein report the first gapless, near-finished genome sequence of the yeast strain Saccharomyces cerevisiae N85 for Chinese rice wine production. Several assembly methods were used to integrate Pacific Bioscience (PacBio) and Illumina sequencing data to achieve high-quality genome sequencing of the strain. The genome encodes more than 6,000 predicted proteins, and 238 long non-coding RNAs, which are validated by RNA-sequencing data. Moreover, our annotation predicts 171 novel genes that are not present in the reference S288c genome. We also identified 65,902 single nucleotide polymorphisms and small indels, many of which are located within genic regions. Dozens of larger copy-number variations and translocations were detected, mainly enriched in the subtelomeres, suggesting these regions may be related to genomic evolution. This study will serve as a milestone in studying of Chinese rice wine and related beverages in China and in other countries. It will help to develop more scientific and modern fermentation processes of Chinese rice wine, and explore metabolism pathways of desired and harmful components in Chinese rice wine to improve its taste and nutritional value.© The Author(s) 2018. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

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September 22, 2019

Biosynthesis of antibiotic chuangxinmycin from Actinoplanes tsinanensis.

Chuangxinmycin is an antibiotic isolated from Actinoplanes tsinanensis CPCC 200056 in the 1970s with a novel indole-dihydrothiopyran heterocyclic skeleton. Chuangxinmycin showed in vitro antibacterial activity and in vivo efficacy in mouse infection models as well as preliminary clinical trials. But the biosynthetic pathway of chuangxinmycin has been obscure since its discovery. Herein, we report the identification of a stretch of DNA from the genome of A. tsinanensis CPCC 200056 that encodes genes for biosynthesis of chuangxinmycin by bioinformatics analysis. The designated cxn cluster was then confirmed to be responsible for chuangxinmycin biosynthesis by direct cloning and heterologous expressing in Streptomyces coelicolor M1146. The cytochrome P450 CxnD was verified to be involved in the dihydrothiopyran ring closure reaction by the identification of seco-chuangxinmycin in S. coelicolor M1146 harboring the cxn gene cluster with an inactivated cxnD. Based on these results, a plausible biosynthetic pathway for chuangxinmycin biosynthesis was proposed, by hijacking the primary sulfur transfer system for sulfur incorporation. The identification of the biosynthetic gene cluster of chuangxinmycin paves the way for elucidating the detail biochemical machinery for chuangxinmycin biosynthesis, and provides the basis for the generation of novel chuangxinmycin derivatives by means of combinatorial biosynthesis and synthetic biology.

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September 22, 2019

Functional genomics of lipid metabolism in the oleaginous yeast Rhodosporidium toruloides.

The basidiomycete yeast Rhodosporidium toruloides (also known as Rhodotorula toruloides) accumulates high concentrations of lipids and carotenoids from diverse carbon sources. It has great potential as a model for the cellular biology of lipid droplets and for sustainable chemical production. We developed a method for high-throughput genetics (RB-TDNAseq), using sequence-barcoded Agrobacterium tumefaciens T-DNA insertions. We identified 1,337 putative essential genes with low T-DNA insertion rates. We functionally profiled genes required for fatty acid catabolism and lipid accumulation, validating results with 35 targeted deletion strains. We identified a high-confidence set of 150 genes affecting lipid accumulation, including genes with predicted function in signaling cascades, gene expression, protein modification and vesicular trafficking, autophagy, amino acid synthesis and tRNA modification, and genes of unknown function. These results greatly advance our understanding of lipid metabolism in this oleaginous species and demonstrate a general approach for barcoded mutagenesis that should enable functional genomics in diverse fungi.

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September 22, 2019

A combinatorial approach to synthetic transcription factor-promoter combinations for yeast strain engineering.

Despite the need for inducible promoters in strain development efforts, the majority of engineering in Saccharomyces cerevisiae continues to rely on a few constitutively active or inducible promoters. Building on advances that use the modular nature of both transcription factors and promoter regions, we have built a library of hybrid promoters that are regulated by a synthetic transcription factor. The hybrid promoters consist of native S. cerevisiae promoters, in which the operator regions have been replaced with sequences that are recognized by the bacterial LexA DNA binding protein. Correspondingly, the synthetic transcription factor (TF) consists of the DNA binding domain of the LexA protein, fused with the human estrogen binding domain and the viral activator domain, VP16. The resulting system with a bacterial DNA binding domain avoids the transcription of native S. cerevisiae genes, and the hybrid promoters can be induced using estradiol, a compound with no detectable impact on S. cerevisiae physiology. Using combinations of one, two or three operator sequence repeats and a set of native S. cerevisiae promoters, we obtained a series of hybrid promoters that can be induced to different levels, using the same synthetic TF and a given estradiol. This set of promoters, in combination with our synthetic TF, has the potential to regulate numerous genes or pathways simultaneously, to multiple desired levels, in a single strain.© 2017 The Authors. Yeast published by John Wiley & Sons, Ltd.

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September 22, 2019

Enhancing the adaptability of the deep-sea bacterium Shewanella piezotolerans WP3 to high pressure and low temperature by experimental evolution under H2O2 stress.

Oxidative stresses commonly exist in natural environments, and microbes have developed a variety of defensive systems to counteract such events. Although increasing evidence has shown that high hydrostatic pressure (HHP) and low temperature (LT) induce antioxidant defense responses in cells, there is no direct evidence to prove the connection between antioxidant defense mechanisms and the adaptation of bacteria to HHP and LT. In this study, using the wild-type (WT) strain of a deep-sea bacterium, Shewanella piezotolerans WP3, as an ancestor, we obtained a mutant, OE100, with an enhanced antioxidant defense capacity by experimental evolution under H2O2 stress. Notably, OE100 exhibited better tolerance not only to H2O2 stress but also to HHP and LT (20 MPa and 4°C, respectively). Whole-genome sequencing identified a deletion mutation in the oxyR gene, which encodes the transcription factor that controls the oxidative stress response. Comparative transcriptome analysis showed that the genes associated with oxidative stress defense, anaerobic respiration, DNA repair, and the synthesis of flagella and bacteriophage were differentially expressed in OE100 compared with the WT at 20 MPa and 4°C. Genetic analysis of oxyR and ccpA2 indicated that the OxyR-regulated cytochrome c peroxidase CcpA2 significantly contributed to the adaptation of WP3 to HHP and LT. Taken together, these results confirmed the inherent relationship between antioxidant defense mechanisms and the adaptation of a benthic microorganism to HHP and LT.IMPORTANCE Oxidative stress exists in various niches, including the deep-sea ecosystem, which is an extreme environment with conditions of HHP and predominantly LT. Although previous studies have shown that HHP and LT induce antioxidant defense responses in cells, direct evidence to prove the connection between antioxidant defense mechanisms and the adaptation of bacteria to HHP and LT is lacking. In this work, using the deep-sea bacterium Shewanella piezotolerans WP3 as a model, we proved that enhancement of the adaptability of WP3 to HHP and LT can benefit from its antioxidant defense mechanism, which provided useful insight into the ecological roles of antioxidant genes in a benthic microorganism and contributed to an improved understanding of microbial adaptation strategies in deep-sea environments.

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